During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. This machinery is driven by E. coli RNA polymerase, which, like all bacterial polymerases, is composed of a core of subunits (beta, beta', alpha1, alpha2, and omega) that have RNA synthesizing activity and a specificity factor (sigma). The sigma protein identifies the start of transcription by recognizing and binding to sequence elements within promoter DNA. During exponential growth, the primary sigma of E. coli is sigma70, which, like all primary sigmas, is composed of four regions. Sigma70 recognizes DNA elements around positions -10 and -35 of host promoter DNA, using residues in its central portion (regions 2 and 3) and C-terminal portion (region 4), respectively. In addition, residues within region 4 must also interact with a structure within core polymerase, called the beta-flap, to position sigma70 region 4 so it can contact the -35 DNA. T4 takes over E. coli RNA polymerase through the action of phage-encoded factors that interact with polymerase and change its specificity for promoter DNA. Early T4 promoters, which have -10 and -35 elements that are similar to that of the host, are recognized by sigma70 regions 2 and 4, respectively. However, although T4 middle promoters have an excellent match to the sigma70 -10 element, they have a phage element (a MotA box) centered at -30 rather than the sigma70 -35 element. Two T4-encoded proteins, a DNA-binding activator (MotA) and a T4-encoded co-activator (AsiA), are required to activate the middle promoters. AsiA alone inhibits transcription from a large class of E. coli promoters by binding to and structurally remodeling sigma70 region 4, preventing its interaction with the -35 element and with the beta-flap. In addition to its inhibitory activity, AsiA-induced remodeling is proposed to make a surface accessible for MotA to bind to sigma70 region 4 in a process called sigma appropriation. Although structures for the N-terminal (NTD) and C-terminal (CTD) of MotA are available, no structures exist for full length MotA or any portion of MotA with DNA. We generated a molecular map of MotA with its DNA element by conjugating single, specific cysteines within MotA with the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE) and determining the FeBABE cut sites obtained with the MotA/AsiA/RNA polymerase/DNA complex. Our work reveals a novel activator/DNA interaction. The MotACTD locates within the 3' portion of the MotA box sequence, while the C-terminal end of the MotANTD and the NTD/CTD linker align with the 5' portion of the MotA box element. Using surface plasmon resonance, we show that sequences both upstream and downstream of the MotA box sequence are needed for optimal binding and that MotA alone very quickly dissociates from the DNA. Our results suggest a model whereby MotA rapidly samples the DNA and is only stably bound once it can also engage its protein partner, the AsiA-remodeled sigma70 present within RNA polymerase.